Purpose :
Long non coding RNAs (lncRNAs) have been reported to regulate autophagy in several biological processes. Studies showed that autophagy is essential in lens organelle degradation and in maintaining lens homeostasis. However, the involvement of autophagy associated lncRNAs in lens is still elusive. We established an efficient system—“fried egg” method in vitro for lentoid body (LB) generation from human pluripotent stem cells (PSCs) during which human lens development in vivo is partially mimicked (ARVO 2015, 2016 and IOVS revised). Thus, this study is to seek potential autophagy associated lncRNAs and their relative mechanisms in human lens development.

Methods :
LBs were derived from human induced PSCs (iPSCs) using “fried egg” method. Expression of LC3B was examined in mouse embryonic lens and LBs using western blot and immunofluorescence assay. Autophagosome was analyzed by transmission electron microscope. Transcriptome of LBs versus iPSCs was examined through microarray analysis. Expressions of selected lncRNAs were verified and further analyzed in human embryonic lens, cataract patients’ capsules and human lens epithelial cells (HLECs) by real-time PCR. Finally, siRNA was used to investigate the function of lncRNA p10540 during autophagy process in HLECs.

Results :
Expression pattern of LC3B in LBs on D18 and D21 was similar to those in mouse lens on E15.5 and P0, implicating that the autophagic activity during LB differentiation mimicked the situation in lens development in vivo. Typical autophagosome was observed in differentiating LBs. Among massive lncRNAs expressed with significant difference between iPSCs and LBs, lncRNA 10540, which was predicted to have co-relationship with the autophagy marker LC3B, highly expressed in differentiating lens fibers in LBs. This result was consistent with its high expression in human embryonic lens on W25-28 and in rapamycin treated HLECs, compared to those in ESCs and untreated HLECs. Furthermore, lncRNA p10540 knockdown resulted in downregulation of LC3B, therefore inhibited autophagy process in HLCEs. Finally, lncRNA p10540 decreased along with age in human lens capsules, indicating its role in age-related cataracts.